Aggregation in antibody therapeutics is a topic of increasing concern in the biopharmaceutical industry, as these aggregates are believed to play an undesirable role resulting in adverse health effects to patients by inducing immunogenicity, altering therapeutic pharmacokinetics, or decreasing efficacy of the drug product. While methods are available to count (and in some cases, image) visible, and to a lesser extent sub-visible, particles, there is currently no reliable way to count and identify aggregates with sizes smaller than 1 micron. Studies on the mechanism of antibody aggregation indicate a process of formation that begins with very small sub-micron sized seed aggregates, which suggests the value of sub-micron characterization as a predictive indicator of therapeutic stability. It is hypothesized that humanized antibody therapeutics break B-cell tolerance by presenting the self-antigen in a foreign array through ordered packing in aggregates; not unlike the mechanism by which viruses elicit a vigorous immune response. This poses the question-what are the cutting-edge technologies utilized by scientists to study the size, shape, and morphology of viruses, and are these technologies applicable to the problem of sub-micron aggregate characterization? Molecular microscopy is a non-invasive molecular imaging technology that uses advanced specimen preparation and imaging methods designed specifically to visualize complex biological samples, under conditions close to their native state. For well-ordered samples such as viruses, and virus-antibody complexes, the achievable resolution can be <0.4 nm. In order to address the problem of quantitative sub- micron aggregate characterization of monoclonal antibody therapeutics, our technology applies high- throughput molecular microscopy to directly visualize antibody monomers and aggregates at multiple scales of magnification to provide quantitative measures of antibody aggregate count, size, shape, and morphology as well as antibody monomer loss in a single experiment. This technology utilizes a complex and expensive TEM instrument and automated software that requires a significant level of expertise to achieve reliable and high- throughput results, and is thus not readily accessible as an in-house method. We propose to make this technology available by incorporating a robust Antibody Therapeutic Submicron Aggregate Characterization service into the armamentarium of fee-for-service TEM capabilities that we already offer to the biopharmaceutical industry. To this end, our Phase II proposal has the following milestones: 1) Defining technology specifications and limitations in quantitative terms that are meaningful to antibody therapeutic formulations and characterization scientists, in consultation with protein therapeutic characterization expert, Dr. Kogan Bao (Allergan Pharmaceuticals, CA); 2) Validating the technology through comparison to accepted methodologies, in partnership with protein aggregation expert, Dr. John Carpenter (UC Denver, CO); and 3) Developing and implementing software algorithms to enhance technology throughput in a cost-effective manner. This will prepare us for Phase III, when we will provide trial service offerings to a select number of our existing pharmaceutical and biotechnology clients. PUBLIC HEALTH RELEVANCE: Aggregation in antibody therapeutics is a topic of increasing concern in the biopharmaceutical industry, because these aggregates are believed to play an undesirable role resulting in adverse health effects to patients; as a result, there is increasing pressure from the U.S. Food and Drug Administration to develop quantitative characterization tools for submicron aggregate particles in biopharmaceutical drug products. Our technology applies high-throughput molecular microscopy to identify and quantify sub-micron protein aggregates in antibody therapeutics. These new analytical methods will contribute to the armamentarium of analytical techniques aimed at helping reduce costs and improve safety and efficacy of antibody therapeutics.